The present invention relates to N-(aroyl)glycine hydroxamic acid derivatives and related compounds that are selective inhibitors of phosphodiesterase (PDE) type IV or of the production of tumor necrosis factor (TNF) and as such are useful in the treatment of asthma, arthritis, bronchitis, chronic obstructive airways disease, psoriasis, allergic rhinitis, dermatitis and other inflammatory diseases as well as AIDS, sepsis, septic shock, cachexia, and other diseases involving the production of TNF. The compounds of this invention may have combined PDE type IV and TNF inhibitory activity. The present invention also relates to the use of such compounds in the treatment of the above diseases in mammals, particularly humans, and to pharmaceutical compositions useful therefor.
Since the recognition that cyclic AMP is an intracellular second messenger (E. W. Sutherland, and T. W. Rall, Pharmacol. Ref., 1960, 12, 265), inhibition of the phosphodiesterases has been a target for modulation and, accordingly, therapeutic intervention in a range of disease processes. More recently, distinct classes of PDE have been recognized (J. A. Beavo and D. H. Reifsnyder, TIPS, 1990, 11, 150), and their selective inhibition has led to improved drug therapy (C. D. Nicholson, R. A. Challiss and M. Shahid, TIPS, 1991, 12, 19). More particularly, it has been recognized that inhibition of PDE type IV can lead to inhibition of inflammatory mediator release (M. W. Verghese et al., J. Mol. Cell Cardiol., 1989, 12, (Suppl. II), S 61) and airway smooth muscle relaxation (T. J. Torphy in Directions for New Anti-Asthma Drugs, eds S. R. O""Donnell and C. G. A. Persson, 1988, 37, Birkhauser-Verlag). Thus, compounds that inhibit PDE type IV, but which have poor activity against other PDE types, inhibit the release of inflammatory mediators and relax airway smooth muscle without causing cardiovascular effects or antiplatelet effects.
TNF is recognized to be involved in many infectious and auto-immune diseases, including cachexia (W. Friers, FEBS Letters, 1991, 285, 199). Furthermore, it has been shown that TNF is the prime mediator of the inflammatory response seen in sepsis and septic shock (C. E. Spooner et al., Clinical Immunology and Immunopathology, 1992, 62, S 11).
The invention relates to compounds of formula 
or a pharmaceutically acceptable salt thereof, wherein
R1 is selected from the group consisting of methyl, ethyl, difluoromethyl and trifluoromethyl;
R2 is (C1-C6)alkyl, (C3-C7)alkoxy(C2-C4)alkyl, phenoxy(C2-C6)alkyl, (C3-C7)cycloalkyl, (C6-C9)polycycloalkyl, phenyl(C1-C8)alkyl or indanyl wherein the alkyl portion of said R2 groups is optionally substituted with one or more fluorine atoms and the aromatic portion of said R2 groups is optionally substituted with one or more substituents independently selected from the group consisting of (C1-C4)alkyl, (C1-C4)alkoxy and halogen;
AA is (AA-1) or (AA-2) wherein:
(AA-1) is 
xe2x80x83wherein R3 and R4 are independently selected from the group consisting of hydrogen, trifluoromethyl, (C1-C6)alkyl, xe2x80x94(CH2)nCO2H, xe2x80x94(CH2)nCONH2, xe2x80x94(CH2)nphenyl, xe2x80x94(CH2)xOH, and xe2x80x94(CH2)xNH2, wherein x ranges from 1 to 5, n ranges from 0 to 5, R5 is hydrogen, OH or (C1-C6)alkyl, and m ranges from 0 to 5; and,
(AA-2) is 
xe2x80x83wherein p ranges from 1 to 4; and,
Y is NHOH or OH.
The term xe2x80x9calkylxe2x80x9d, as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight or branched moieties.
The term xe2x80x9calkoxyxe2x80x9d, as used herein, includes O-alkyl groups wherein xe2x80x9calkylxe2x80x9d is defined above.
The term xe2x80x9ccycloalkylxe2x80x9d, as used herein, includes saturated monovalent cyclo hydrocarbon radicals including cyclobutyl, cyclopentyl and cycloheptyl.
The term xe2x80x9cpolycycloalkylxe2x80x9d, as used herein, includes saturated monovalent polycyclo radicals comprising ring assemblies that are fused, bicyclo or tricyclo. Such ring assemblies include bicycloheptyl, bicyclobutyl, tricyclooctanyl and perhydropentalenyl.
The term xe2x80x9carylxe2x80x9d, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl, optionally substituted by 1 to 3 substituents selected from the group consisting of fluoro, chloro, trifluoromethyl, (C1-C6)alkoxy, (C6-C10)aryloxy, trifluoromethoxy, difluoromethoxy and (C1-C6)alkyl.
The term xe2x80x9ctreatmentxe2x80x9d as used herein, unless otherwise indicated, includes (i) methods to cure, relieve or lessen the undesirable effects of, or the undesirable symptoms associated with, conditions and diseases that respond to the inhibition of PDE type IV or the inhibition of the production of TNF, where such conditions and diseases are actively occurring in a mammal, including a human, and (ii) methods to prevent such conditions and diseases from occurring in a mammal, and (iii) methods to slow the onset of such conditions and diseases in a mammal. The terms xe2x80x9ctreatxe2x80x9d and xe2x80x9ctreatingxe2x80x9d as used herein are defined in accord with the above definition.
The term xe2x80x9ctherapeutically effective amountxe2x80x9d as used herein, unless otherwise indicated, means an amount effective to inhibit PDE type IV or inhibit the production of TNF, or an amount effective in the treatment, as defined above, of a condition or disease that responds to the inhibition of PDE type IV or the inhibition of the production of TNF.
The compounds of formula I include certain compounds having chiral centers which therefore exist in different enantiomeric forms, This invention relates to all optical isomers and stereoisomers of the compounds of formula I and mixtures thereof.
Preferred compounds of formula I include those in which R1 is methyl.
Other preferred compounds of formula I include those in which R2 is cyclopentyl.
Other preferred compounds of formula I include those in which AA is the moiety (i) and R3 is hydrogen, methyl, trifluoromethyl or xe2x80x94CH2OH.
Other preferred compounds of formula I include those in which AA is the moiety (AA-1) and R4 is hydrogen.
Other preferred compounds of formula I include those in which AA is the moiety (AA-1) and R5 is hydrogen.
Other preferred compounds of formula I include those in which AA is the moiety (AA-1) and m is 0.
Other preferred compounds of formula I include those in which Y is xe2x80x94NHOH.
Specific preferred compounds of formula I include the following:
xcex1-monofluoromethyl-xcex1-N-[(3-cyclopentyloxy-4-methoxy)benzoyl]glycine hydroxamic acid;
xcex1-difluoromethyl-xcex1-[(3-cyclopentyloxy-4-methoxy)benzoyl]glycine hydroxamic acid;
xcex1-ethyl-xcex1N-[(3-cyclopentyloxy-4-methoxy)benzoyl]glycine hydroxamic acid;
xcex1-propyl-xcex1-N-[(3-cyclopentyloxy-4-methoxy)benzoyl]glycine hydroxamic acid;
xcex1-N-[(3-cyclopentyloxy-4methoxy)benzoyl]-D-cystine hydroxamic acid;
xcex1-trifuoromethyl-xcex1-N-[(3-cyclopentyloxy-4-methoxy)benzoyl]glycine hydroxamic acid;
xcex1-N-[(3-cyclopentyloxy-4-methoxy)benzoyl]-D-serine hydroxamic acid;
xcex1-N-[(3-cyclopentyloxy-4-methoxy)benzoyl]glycine hydroxamic acid; and,
xcex1-N-[(3-cyclopentyloxy-4-methoxy)benzoyl]-D-alanine hydroxamic acid.
The present invention further relates to a pharmaceutical composition for the inhibition of PDE type IV or the inhibition of the production of TNF in a mammal, including a human, comprising a therapeutically effective amount of a compound according to formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
The present invention further relates to a pharmaceutical composition for the treatment of a condition or disease selected from the group consisting of asthma, arthritis, bronchitis, chronic obstructive airways disease, psoriasis, allergic rhinitis, dermatitis, AIDS, septic shock and other conditions or diseases that respond to the inhibition of PDE type IV or the inhibition of the production of TNF in a mammal, including a human, comprising a therapeutically effective amount of a compound according to formula I or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.
The present invention further relates to a method of inhibiting PDE type IV or inhibiting the production of TNF in a mammal, comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound according to formula I or a pharmaceutically acceptable salt thereof.
The present invention further relates to a method of treating a condition or disease selected from the group consisting of asthma, arthritis, bronchitis, chronic obstructive airways disease, psoriasis, allergic rhinitis, dermatitis, AIDS, septic shock and other conditions or diseases that respond to the inhibition of PDE type IV or the inhibition of TNF in a mammal, including a human, comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound according to formula I or a pharmaceutically acceptable salt thereof.
The following reaction Scheme I illustrates the preparation of the compounds of the present invention. Unless otherwise indicated, R1, R2, R3, R4, R5, AA, n, m, p, and Y, as used in Scheme 1 and the following discussion, are as defined above. In Scheme 1 and the Preparations and Examples that follow, all synthesis reactions and other procedures are done at room temperature (20-25xc2x0 C.) unless otherwise indicated. 
In reaction 1 of Scheme 1, a carboxylic acid of formula V is coupled to O-benzylhydroxylamine to obtain a compound of formula VI using a coupling method well known to those skilled In the art of peptide chemistry. The carboxylic acid of formula V is available from various commercial sources or can be prepared according to synthetic methods known to those skilled in the art. The preferred coupling method is to combine the compound of formula V with O-benzylhydroxylamine hydrochloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a base, such as triethylamine, in an inert solvent, such as methylene chloride, at a temperature of 0xc2x0 C. to 30xc2x0 C. (20-25xc2x0 C. preferred) for a period of 2 hours to 48 hours (16 hours preferred).
In reaction 2 of Scheme 1, the compound of formula VI is treated with an acid, such as hydrochloric acid or trifluoroacetic acid, to remove the t-butyloxycarbonyl group to give a salt of formula VII, wherein X of HX is chloride or trifluoroacetate, and m, R3, R4 and R5 are as defined above.
In reaction 3 of Scheme 1, the salt of formula VII is coupled to a benzoic acid derivative of formula VIII to prepare the compound of formula IX using a coupling method well known to those skilled in the art of peptide chemistry. The benzoic acid derivative of formula VIII can be prepared according to synthetic methods known to those skilled in the art. For instance, 3-cyclopentyloxy-4-methoxybenzoic acid can be prepared according to the method described in M. N. Palfreyman et al., J. Med. Chem., vol. 37, page 1696 (1994), which is herein incorporated by reference. The preferred coupling method is to combine the compound of formula VIII with the salt of formula VII, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and a base, such as triethylamine, in an inert solvent, such as methylene chloride, at a temperature of 0xc2x0 C. to 30xc2x0 C. (20-25xc2x0 C. preferred) for a period of 2 hours to 48 hours (16 hours preferred).
In reaction 4 of Scheme 1, the compound of formula IX is hydrogenated over Pd(OH)2 in a solvent such as methanol or ethanol for a period of 4 to 48 hours (16 hours preferred) to obtain the compound of formula X.
While Scheme 1 illustrates the preparation of compounds of formula I wherein AA is (AA-1), the preparation of compounds of formula I wherein AA is (AA-2) follows essentially the same route. In particular, in the first step, the compound of formula V is replaced with a compound of the same formula except the AA portion is (AA-2) rather than (AA-1). Such compounds are commercially available or can be made by synthetic techniques known to those skilled in the art. Then, reactions 1-4 are performed as described above.
To prepare compounds of formula I wherein Y is OH rather NHOH, the process begins at the third reaction of Scheme 1 where the compound of formula VII is replaced with a compound of formula XI: HX.NHR5CR3R4(CH2)mCO2CH2Ph. Compounds of formula XI are commercially available or can be made by synthetic techniques known to those skilled in the at. The compound of formula XI is coupled with the compound of formula VIII as described above for reaction 3 of Scheme 1. Then, reaction 4 of Scheme 1 is followed as described above to prepare the compound of formula I wherein Y is OH.
Pharmaceutically acceptable acid addition salts of the compounds of this invention include, but are not limited to, those formed with acetic, lactic, succinic, maleic, tartaric, citric, gluconic, ascorbic, benzoic, cinnamic fumaric, sulfuric, phosphoric, hydrochloric, hydrobromic, toluenesulfonic, mandelic, di-p-toluoyl-1-tartaric and related acids. The acid addition salts of the compounds of formula I are prepared in a conventional manner by treating a solution or suspension of the free base of formula I with about one chemical equivalent of a pharmaceutically acceptable acid. Conventional concentration or crystallization techniques are employed in isolating the salts. Pharmaceutically acceptable cationic salts of the compounds of formula I wherein Y is hydroxyl include, but are not limited to, those of sodium, potassium, calcium, magnesium, ammonium, N,Nxe2x80x2-dibenzylethylenediamine, N-methylglucamine (meglumine), ethanolamine and diethanolamine.
The ability of the compounds of formula I and their pharmaceutically acceptable salts to inhibit PDE type IV or inhibit the production of TNF and, consequently, demonstrate their effectiveness for treating diseases that respond to the inhibition of PDE IV or the inhibition of the production of TNF is shown by the following in vitro assay tests.
Human peripheral blood is collected in ethylenediaminetetraacetic acid, diluted 1:2 in piperazine-N,Nxe2x80x2-bis-2-ethanesulfonic acid (PIPES) buffer and then layered over percoll solution. Gradients are formed by centrifugation for 30 minutes at 2000 rpm at 40xc2x0 C. The remainder of the isolation procedure, which is based on the procedure of Kita et al., J. Immunol., 152, 5457 (1994), is carried out at 4xc2x0 C. The neutrophil/eosinophil layer is collected from the percoll gradient and the red blood cells are lysed. Remaining cells are washed in PIPES (1% FCS), incubated with anti-CD16 microbeads (MACS) for 1 hour, and passed over a magnetic column to remove the neutrophils. Eosinophils are collected in the eluate and analyzed for viability by trypan blue and purity by diff-quick stain. Eosinophil purity is routinely greater than 99% using this method.
Purified eosinophils are resuspended in 750 xcexcL of PDE lysis buffer (20 mM triethylamine, 1 mM ethylenediaminetetraacetic acid, 100 xcexcg/ml bacitracin, 2 mM benzamidine, 50 xcexcM leupeptin, 50 xcexcM PMSF, 100 xcexcg/ml soybean trypsin inhibitor) and quick frozen in liquid nitrogen. Cells are thawed slowly and sonicated. Membranes are vortexed (disruption is confirmed by Trypan blue staining of fragments). Disrupted cells are centrifuged at 45 k rpm for 30 minutes at 4xc2x0 C. to isolate membranes. Cytosol is decanted, and membrane resuspended to 200 xcexcg/ml for use as PDE source in the hydrolysis assay yielding a window from 3000 to 5000 counts.
Compounds are dissolved in dimethyl sulfoxide at 10-2M, then diluted 1:25 in water to 4xc3x9710xe2x88x924 M. This suspension is serially diluted 1:10 in 4% dimethyl sulfoxide, for a final dimethyl sulfoxide concentration in the assay of 1%.
To 12xc3x9775 mm glass tubes add:
25 xcexcl PDE assay buffer (200 mM Tris/40 mM MgC12)
24 xcexcl 4 nM/ml CAMP stock
25 xcexcl test compound
25 xcexcl PDE source (membrane)
Background control=membrane boiled 10xe2x80x2
Positive controlxe2x80x9425 xcexcl unboiled membrane
Incubate 25 minutes in 37xc2x0 C. water bath.
Reaction is stopped by boiling samples 5 minutes. Samples are applied to Afri-gel column (1 ml bed volume) previously equilibrated with 0.25 M acetic acid followed by 0.1 mM N-[2-hydroxyethyl]piperazine-Nxe2x80x2-2-ethanesulfonic acid (HEPES)/0.1 mM NaCl wash buffer (pH 8.5). cAMP is washed off column with HEPES/NaCl, 5xe2x80x2-AMP is eluted in 4 ml volumes with 0.25 M acetic acid. 1 ml of eluate is counted in 3 ml scintillation fluid for 1 minute ([3H].
Substrate conversion=(cpm positive controlxc3x974)/total activity. Conversion rate must be between 3 and 15% for experiment to be valid.
% Inhibitionxe2x88x921-(eluted cpmxe2x88x92background cpm/control cpmxe2x88x92bkgd cpm)xc3x97100.
IC50 values are generated by linear regression of inhibition titer curve (linear portion); and are expressed in xcexcM.
The ability of the compounds of formula I and the pharmaceutically acceptable salts thereof to inhibit the production of TNF and, consequently, demonstrate their effectiveness for treating diseases involving the production of TNF is shown by the following in vitro assay:
Peripheral blood (100 mls) from human volunteers is collected in ethylenediaminetetraacetic acid (EDTA). Mononuclear cells are isolated by Ficoll/Hypaque and washed three times in incomplete Hanks"" balanced salt solution (HBSS). Cells are resuspended in a final concentration of 1xc3x97106 cells per ml in prewarmed RPMI (containing 5% FCS, glutamine, pen/step and nystatin). Monocytes are plated as 1xc3x97106 cells in 1.0 ml in 24-well plates. The cells are incubated at 37xc2x0 C. (5% carbon dioxide) and allowed to adhere to the plates for 2 hours, after which time non-adherent cells are removed by gently washing. Test compounds (10 xcexcl) are then added to the cells at 3-4 concentrations each and incubated for 1 hour. Lipopolysaccharide (LPS) (10 xcexcl) is added to appropriate wells. Plates are incubated overnight (18 hrs) at 37xc2x0 C. At the end of the incubation period TNF was analyzed by a sandwich ELISA (RandD Quantikine Kit). IC50 determinations are made for each compound based on linear regression analysis.
For administration to humans in the curative or prophylactic treatment of inflammatory diseases, oral dosages of the compounds of formula I and the pharmaceutically acceptable salts thereof (hereinafter also referred to as the active compounds of the present invention) are generally in the range of from 0.1-400 mg daily for an average adult patient (70 kg). Thus for a typical adult patient, individual tablets or capsules contain from 0.1 to 50 mg of active compound, in a suitable pharmaceutically acceptable vehicle or carrier. Dosages for intravenous administration are typically within the range of 0.1 to 40 mg per single dose as required. For intranasal or inhaler administration, the dosage is generally formulated as a 0.1 to 1% (w/v) solution. In practice the physician will determine the actual dosage which will be most suitable for an individual patient and it will vary with the age, weight and response of the particular patient. The above dosages are exemplary of the average case but there can, of course, be individual instances where higher or lower dosage ranges are merited, and all such dosages are within the scope of this invention.
For administration to humans for the inhibition of TNF, a variety of conventional routes can be used including oral, parenteral and topical administration routes. In general, the active compound will be administered orally or parenterally at dosages between about 0.1 and 25 mg/kg body weight of the subject to be treated per day, preferably from about 0.3 to 5 mg/kg. The compound of formula I can also be administered topically in an ointment or cream in concentrations of about 0.5% to about 1%, generally applied 2 or 3 times per day to the affected area. However, some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
For human use, the active compounds of the present invention can be administered alone, but will generally be administered in an admixture with a pharmaceutical diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsules or ovales either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. They may be injected parenterally; for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other substances; for example, enough salts or glucose to make the solution isotonic.